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Three-dimensional scaffold of electrosprayed fibers with large pore size for tissue regeneration
Acta Biomaterialia 6 (2010) 4734–4742
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Three-dimensional scaffold of electrosprayed fibers with large pore size for tissue regeneration Jong Kyu Hong, Sundararajan V. Madihally * School of Chemical Engineering, Oklahoma State University, 423 Engineering North, Stillwater, OK 74078, USA
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Article history: Received 22 April 2010 Received in revised form 30 June 2010 Accepted 2 July 2010 Available online 8 July 2010 Keywords: Electrospraying PCL Gelatin Tissue regeneration Porous structure
a b s t r a c t The regeneration of tissues using biodegradable porous scaffolds has been intensely investigated. Since electrospinning can produce scaffolds mimicking nanofibrous architecture found in the body, it has recently gained widespread attention. However, a major problem is the lack of pore size necessary for infiltration of cells into the layers below the surface, restricting cell colonization to the surfaces only. This study describes a novel twist to the traditional electrospinning technology: specifically, collector plates are designed which allow the formation of very thin layers with pore sizes suitable for cell infiltration. The thin samples could be handled without mechanically damaging the structure and could be transferred into cell culture. These thin layers were stacked layer-by-layer to develop thick structures. Thirty day cultures of fibroblasts show attachment and spreading of cells in every layer. This concept is useful in regenerating thick tissues with uniformly distributed cells and others in in vitro cell culture. Ó 2010 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.
1. Introduction Tissue engineering or regeneration focuses on restoring, maintaining and enhancing tissue and organ function [1]. In tissue engineering, biodegradable scaffolds are used to support and guide cells to proliferate, organize and produce their own extracellular matrix (ECM) [2] so that, when the scaffolding material eventually disappears, only the necessary healthy tissue is left in a topologically required form [3]. A biomimic matrix for growing cells is a fundamental element for tissue regeneration. Templates generated from natural [4] and synthetic polymers or after removing the cellular components from xenogeneic tissues [5] have been used to support and guide the in-growth of cells. Synthesizing scaffolds using techniques such as controlled rate freezing and lyophilization [6], the porogen-leaching technique [7], the gas-foaming technique [8], microfabrication [9] and free-form printing [10] has attracted increasing attention. Although each technique has advantages and disadvantages, the electrospinning technique has recently emerged as a novel technique for tissue regeneration because it allows fabrication of nano- and micrometer size fibers, which are very similar to fibers in the natural extracellular environment. The process of fabricating nano- to micrometer synthetic fibers with non-woven structures using electrostatic forces, called electrospinning (electrospray and spinning), has been known for over * Corresponding author. Tel.: +1 405 744 9115; fax: +1 405 744 6338. E-mail addresses: [email protected], [email protected] (S.V. Madihally).
100 years. Although electrospinning technology was first patented in the US in 1902 [11], this technology was latent until the era of nanoscience and nanotechnology in the 1990s. The electrospinning process is versatile and its productivity is higher than other methods of fabrication of nanofibers. With a suitable polymer and appropriate solvent system, fiber size can be controlled (from 40 nm to 10 lm) by manipulating the process parameters. Since the technology allows the possibility of tailoring the mechanical and biological properties, there significant efforts have been made to adapt the technology for use in tissue regeneration [12,13]. Most soluble polymers with a high molecular weight can be electrosprayed (fibers collected on a stationary collector plate) or electrospun. Nanofibers can be made of natural and synthetic polymers, or polymer blends. A major problem in electrospinning technology is the lack of generating structural features necessary for building three-dimensional (3-D) tissues [12,14]. The pore sizes are less than 10 lm, while human cells are typically greater than 10 lm in size [15]. Hence, cell growth is restricted to the surface only. The reduced pore size is primarily due to multiple layers of fibers deposited to obtain thicker structures that withstand mechanical handling in subsequent steps. Addressing the cell seeding problem by depositing both the polymer and the cells simultaneously is a recent advancement [16]. However, this approach is not practical as cells will be exposed to (i) toxic organic solvents used in the process, (ii) high shear rates induced by the flow of fluids through narrow nozzles and (iii) sub-optimal environmental conditions during manufacturing. These factors affect cell viability and growth function tissue growth. Due to reduced pore size and restricted cell
1742-7061/$ - see front matter Ó 2010 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.actbio.2010.07.003
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infiltration, it is not possible to regenerate tissues of a thickness suitable for clinical transplantation. In this study, we introduce an innovative electrospraying technique with a novel collector plate which allows the formation of very thin layers of micro- and nanofibers. Due to reduced thickness, pore size (60 lm) of the formed layers is suitable for mammalian cell infiltration. We performed the fibroblast cell culture of multiple layers using layer-by-layer assembly: place one layer of thin fibers, seed cells and repeat. We show 3-D scaffolds of thin layers of electrosprayed fibers in which human fibroblasts are growing not only horizontally but also vertically due to the large pore sizes of the electrosprayed fibers. The implications of producing 3-D scaffolds using the layer-by-layer technique can be extrapolated to other cells, fibers, tissues and organs, and could become a phenotype of 3-D scaffolds in tissue regeneration. 2. Materials and methods 2.1. Materials Polycaprolactone (PCL; Mn = 80,000) and gelatin type A were purchased from Sigma (St. Louis, MO) and PCL (Mw = 43,000–50,000) was obtained from Polysciences (Warrington, PA). Chloroform, methanol and acetic acid were purchased from Pharmco (Brookfield, CT). For cell culture study, human foreskin fibroblast (HFF-1) was obtained from the American Type Culture Collection (Walkersville, MD). Dulbecco’s modified Eagle’s medium (DMEM), glucose and amphotericin B were obtained from Sigma (St. Louis, MO). Fetal
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bovine serum (FBS), Alexa Fluor 546 and 40 ,6-diamidino-2-phenylindole (DAPI) were purchased from Invitrogen (Carlsbad, CA). TrypLE Express, penicillin–streptomycin and L-glutamine were obtained from Gibco (Grand Island, NY) and sodium bicarbonate was purchased from EMD (Gibbstown, NJ). 2.2. Fabrication of the thin layer of electrosprayed fibers Electrospraying apparatus consisted of a syringe pump (74,900 series, Cole-Parmer Instrument Company, Vernon Hills, IL), a BD 10 ml syringe (Luer-Lok Tip; Becton Dickinson and Company, Franklin Lakes, NJ), a needle tip, a high-voltage power supply (ES30P-5W/DAM, Gamma high Voltage Research, Ormond Beach, FL), earth grounding and a collector plate (Fig. 1A). The novel collector had four holes (diameter, 1.9 cm and depth 1.2 cm) in a wooden frame (3 10 2 cm) wrapped with aluminum foil (Fig. 1E). A conventional collector plate that was the same size as the novel collector but without a hole was also wrapped with aluminum foil. Electrosprayed fibers were fabricated at 40 ml h1 and 20 kV. For microsized fibers, 20% (w/v) PCL (Mw = 43,000–50,000) dissolved in a mixture solvent of chloroform and methanol (mixing ratio 9 to 1) was sprayed using a 14G needle. The distance of the collector plate was 12 cm from the tip of the needle. For nanosized fibers, 10% (w/v) PCL dissolved in a mixture solvent of chloroform and methanol (mixing ratio 9 to 1) was sprayed using a 26G needle. The distance of the collector plate was 15 cm from the tip of the needle. Deposit volume of the solution was 0.75 ml. Gelatin fibers were formed using 20% (w/v) gelatin dissolved in acetic acid and
Fig. 1. Novel electrospray set-up. (A) Schematic representation of conventional electrospray set-up. (B) Schematic representation of novel electrospray set-up. (C) Photograph of conventional electrospray set-up. (D) Photograph of novel electrospray set-up. (E) Photograph of the novel collector.
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distilled water (mixing ratio 7 to 3). Flow rate, needle size, highvoltage supply and distance between the needle tip and the collector were 10 ml h1, 20G, 10 kV and 8 cm, respectively. For simultaneous and sequential deposit, 20% w/v PCL (Mn = 80,000) was prepared with a solvent mixture of chloroform and methanol (mixing ratio 9 to 1). In the simultaneous and the sequential processes, fibers were formed at the same condition described for gelatin fiber formation using the novel collector. 2.3. Microstructure characterization Fig. 2. Schematic of the process of cell seeding using layer-by-layer assembly.
Samples were analyzed using a JEOL 6360 electron microscope (Jeol USA Inc., Peabody, MA) at an accelerated voltage of 9–20 kV, similar to our previous publications [17]. In brief, samples were attached to an aluminum stub using a conductive graphite glue (Ted Pella Inc., Redding, CA) and sputter-coated with gold for 1 min before scanning electron microscopy (SEM) study. Along with the obtained SEM images, fiber diameters, pore size and shape factors of pores (4p area/perimeter2; the closer the number is to 1, the closer the cell shape is to a circle) were quantified using Sigma Scan Pro (SPSS Science, Chicago, IL). More than 35 points of fibers and pores were analyzed. 2.4. Mechanical test Microsize fibers were fabricated using a collector plate (10 3.5 2 cm) with a rectangular hole (5 1 1 cm) and an additional flat surface area or peripheral area of a rectangular hole, resulting in two rectangular holes (one with a flat surface and the other without). Fibers accumulated on the flat surface were used as samples for conventional fibers while the fibers collected in the hole were used as samples for novel fibers. Tensile testing was performed at room temperature with dry conditions using the method described previously [18]. In brief, 25 mm (width) 10 mm (length) rectangular strips were cut from the novel and conventional samples and strained to break at a constant crosshead speed of 10 mm min1 using INSTRON 5542 tension machine (INSTRON, Canton, MA). 2.5. Cell culture HFF-1 was cultured in DMEM supplemented with 4 mM L-glutamine, 4.5 g l1 glucose, 1.5 g l1 sodium bicarbonate, 100 U ml1 penicillin–streptomycin, 2.5 mg ml1 amphotericin B and 15% FBS. The cells were maintained in a CO2 incubator at 37 °C. The medium was changed every 2 days. Before cell seeding, cells were detached with 2% TrypLE Express, centrifuged and suspended in serum-added medium. Microsize fibers were fabricated using 20% w/v of PCL (Mw = 43,000) in chloroform/methanol (ratio 9 to 1) and the novel collector. Flow rate, needle size, high-voltage supply and distance between the needle tip and the collector were 10 ml h1, 20G, 10 kV and 8 cm, respectively. The deposit volume was 0.75 ml. Fibers on the aluminum frame were sterilized using UV light for 4 h, immersed in ethanol for 4 h and rinsed with phosphate-buffered saline (PBS) three times. The thin layers were soaked in serumadded medium for 4 h. The cell culture plates were treated with albumin to minimize cell adhesion to the tissue culture plastic surface prior to insertion of a thin layer of fibers (Fig. 2). Then 30,000 cells in 100 ll were seeded onto five points of fibers in a single layer of the aluminum frame. For three-layer cultures, the cell culture in the single layer was repeated three times using the layer-by-layer technique: place one layer of the sample, seed cells on five points of the sample and repeat the process of sample loading and cell seeding twice. Ten
minutes after cell seeding, 2 and 3 ml of medium were added to six-well plates for single and triple layers, respectively. 2.6. Evaluating of cell distribution and organization At 1, 4 and 7 days after cell culture, the sample was fixed with 3.7% formaldehyde, rinsed with PBS and immersed in ethanol at 20 °C. The sample was then stained with 2 ml of Alexa phalloidin (0.66 lM) for cytoskeletal actin and counterstained with 2 ml of DAPI (30 nmol) for nuclei of cells, respectively [19]. The samples were first observed under an inverted fluorescence microscope (Nikon TE2000, Melville, NY). Digital micrographs were collected using a CCD camera. The data were treated using Corel Paint Shop Pro Photo X2 to modulate the brightness, contrast and clarification. Also, 3-D images of cells on fibers in the single and triple layers were collected using a confocal microscope (Leica TCS SP II, Heidelberg, Germany). Image stacks of both single and triple layers along the z-axis were collected at a step size of 1.282 lm for the singlelayer cultures and 4.271 lm for the three-layer cultures. The observed thickness of the z-axis for single and triple layers were 30.77 and 89.70 lm, respectively. At each step, the two fluorescent signals of nuclei and skeletal actin and one light signal of fibers were collected. Then fluorescent and light signals of cells on fibers in the single layer at 15.36 lm along the z-axis were overlapped. To obtain a 3-D hologram, fluorescence signals of cells at each step were merged in the three-layer sample. Samples were also analyzed using SEM to evaluate the distribution of fibers and cells. For this purpose, the samples were dried using ethanol and analyzed similar to the fibers described above (Section 2.3). 2.7. Histology assay After 30 days of cell culture, the samples formed using single and triple layers of fibers were tailored using tweezers and scissors. The aluminum frames of the samples were removed completely. Random parts of the samples were fixed in 3.7% formaldehyde, dried through an ethanol gradient and embedded in paraffin. The samples were cut into 4 lm and stained with hematoxylin and eosin (H & E) for histology analysis [20]. 3. Results 3.1. Fabrication of the thin layer of highly porous fibers A conventional electrospray set-up consists of a syringe pump, a syringe and a needle of appropriate size, a high-voltage power supply, earth grounding and a stationary collector plate (Fig. 1A). The collector plate is a flat conducting plate where the polymer fibers accumulate. After a certain elution volume, the fibers are removed and used in the required application. To remove the material, larger volumes are deposited, which leads to fibrous structures with limited pore size.
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We questioned whether the fibers could be accumulated by creating a hole in the collector plate. For this purpose, different size holes were created in wood, plastic or glass, and wrapped around with a conductive aluminum foil (Fig. 1B), which can be autoclaved when sterilization is necessary. The collector plate was designed with the intention of using it as a support structure while transporting and subsequent handling. Experiments performed under the same conditions as the traditional collector plate showed the presence of fibers in the holed area, suggesting the possibility of forming thin layers. Different shapes, such as circle, hexagon and rectangles, of various sizes (up to 10 cm) were tested. These results showed the possibility of obtaining thin layers. Since the aluminum foil could be simply peeled off from the core structure, it is also easy for the thin layer to be handled without mechanical damage from external stress. The absence of a continuous conducting medium could alter the electrical field. Hence, we questioned how the distribution of fibers would be altered and whether the fibers formed would have a different architecture. Similar to other reports, fibers generated by the conventional plate showed multiple layers of fibers (Fig. 3A) under SEM. However, few layers of fibers were observed in the open area of the new collector plate (Fig. 3B), while multiple layers were observed on the sheet. Many fiber morphologies appeared smooth and similar between the two conditions, except in the case of a large fiber; in the conventional plate (Fig. 3A), many fibers showed pores on their surface. To test whether nanosize fibers could be obtained using the new collector plate, processing parameters were optimized by manipulating the flow rate of the solution, the strength of voltage, and the distance between the needle tip and the collector. The conventional collector plate showed multilay-
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ered nanofibers (Fig. 3C). Similar nanofibers were collected on the new collector plate with a single layer (Fig. 3D), suggesting the applicability of this collector plate to other conditions. To test whether the new collector plate is applicable to other polymeric systems, the formation of fibers using naturally derived gelatin was tested. The results showed that the gelatin fibers accumulated similar to the PCL fibers (Fig. 3E). Others have also used sequential (exchanging the syringes containing two different polymeric solutions in sequence) and simultaneous (using two syringes containing two different polymeric solutions at the same time) processes of electrospraying to deposit two different polymers for controlling the microarchitecture [21–24]. To test this possibility, PCL and gelatin were deposited sequentially and simultaneously. The results showed (Fig. 3F) that thin layers of fibers of different polymers could be collected on the novel collector plate. To understand the effect of hydration on the fibers, formed fibers were observed under light microscopy before and after hydration. Upon hydration in PBS, the PCL fiber structure remained the same. However, gelatin fibers dissolved completely in PBS as they were not stabilized by any cross-linker. When composite structures were hydrated, PCL fibers remained intact even after 30 or more days. However, the fibers made of PCL and gelatin were entangled and collapsed within 15 min because gelatin dissolved in PBS. Thus, one could easily understand the alterations in fibrous architecture using these thin layers. 3.2. Fiber characterization To determine the fiber size and pore size, fibers were generated under the same condition of solution (concentration of solution)
Fig. 3. Morphology of fibers using novel and conventional collectors. Microfibers made of PCL (Mw = 43,000) with (A) a conventional collector and (B) a novel collector under the same conditions. Nanofibers made of PCL (Mw = 43,000) with (C) a conventional collector and (D) a novel collector under the same conditions. (E) Fibers made of gelatin type A using the novel collector. (F) Hybrid fibers made of microsize PCL (Mn = 80,000) fibers and nanosize gelatin type A fibers in sequential process using the novel collector.
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and process parameters (flow rate, needle size, distance between the needle tip and the collector, and high-voltage supply) using the novel and the conventional collector plates. Quantification of scanning electron micrographs showed that the new collector plate does not affect the fiber size and the shape of the pores in the scaffolds (Fig. 4A). Even when nanofibers were formed under other conditions, no significant difference was obtained. Thus, this new collector plate can be easily adapted to various other polymers in conjunction with the intended application. Controlling fiber sizes is also possible using appropriate solvents and the distance of the collector plate. This suggests that the technology is versatile and can be easily integrated into existing technologies. The shape factor (Fig. 4B) of pores formed due to random distribution of fibers was similar in the two cases. Pore size (Fig. 4C) of the scaffold formed with the new collector plate showed significant increase due to the decreased density of fibers deposited in the open area. However, under the same conditions of solution and process parameters, the diameters of fibers generated by the novel and conventional fibers were quite similar. The pore size of fiber produced with the novel collector is much greater than those produced with the conventional one. Collected fibrous structures at the same condition for the same time were tested under tensile loading in the dry state at room temperature. Load–extension curves (Fig. 4D) showed that the extension of novel fibers was twice as long as that of conventional fibers; novel fibers showed 250% strain, whereas the conventional fibers showed 100% strain. However, the load-carrying capacity of the fibrous structures was nearly half that of the conventional fibers. These differences could be attributed to the decreased density of fibers in the novel collector plate; due to free space between the fibers on the sample, thin layers can stretch but cannot carry too much load.
3.3. Developing 3-D colonized cells To test whether cells can be cultured on these thin layers, porous structures formed along with the aluminum foil were inserted onto albumin-coated culture plates (to minimize cell adhesion to the tissue culture plastic) seeded with HFF-1 cells. For evaluating layer-by-layer assembly, the process of inserting the thin layer and seeding cells was done three times (Fig. 2). The entire assembly was cultured for 7 days in serum-containing medium with routine monitoring under light microscopy. An increase in the number of cells was observed on fibers in both the single and triple layers. Further, many cells in both the single and triple layers appeared in clusters at the initial time point, but showed spreading and a spindle shape. By day 7, a number of pores were covered with the regenerated components. To assess the cellular components, they were stained for cytoskeletal actin and counterstained for nucleus using DAPI. To avoid background fluorescence signal from cells on the surface of the cell culture dish, the samples were moved into new dishes after the staining and washing. Fluorescent images (Fig. 5) in single or triple layers showed a qualitative improvement in the cells up to 7 days. Moreover, cells were individually distributed and surrounded by matrix elements, suggesting that the cells are functional. In addition, some cells were focused in the plane of view and others were unfocused, as they were attached to different layers of fibers. To better understand the distribution of cells in three dimensions, confocal microscopic analysis was performed using a step size of 1.2821 lm. Reconstructed confocal images of three layers showed the presence of cells in every 89.7 lm thick layer of the three-layer culture (Fig. 6). More cells appear in a single layer culture as they are distributed in a narrow thickness. To assess the distribution of cells in relation to fibers, a light microscopy image
Fig. 4. Comparison of fibrous layer characteristics. (A) Fiber diameters, with error bars corresponding to the standard deviation (n = 50). (B) Shape factor, with error bars corresponding to the standard deviation (n = 50). (C) Box plot showing the distribution of pore size with 10th, 25th, 50th, 75th and 90th percentiles and the mean value (thick line within each box). Values that were outside the 95th and 5th percentiles were treated as outliers. (D) The load–extension behavior.
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Fig. 5. Cell colonization in thin layers using layer-by-layer assembly. (A–C) Fluorescent micrographs of cells on single layer of fibers. (D–F) Fluorescent micrographs of cells on three layers of fibers. The samples were stained with Alexa phalloidin for cytoskeletal actin (red) and counterstained with DAPI for nuclei (blue) of cells. The scale bar corresponds to 50 lm.
was overlayed with fluorescent images in the same plane. The obtained micrograph showed that cells were surrounded by fibers and attached to the fibers, particularly in pores which were not too large relative to the size of the cells. Note that Fig. 6H is a 3-D image which can be better visualized using 3-D spectacles. This suggests that cells are distributed continuously in three dimensions. Samples were also evaluated using SEM to determine the distribution of cells within the fibers. These results revealed (Fig. 7A) that the cells on all fibers were attached to the fibers and entangled
by them. Cells attached and spread throughout the matrix. In some locations, cells showed numerous lamellipodia and filopodia with anchorage points to the fibers (Fig. 7B), probably via the adhesion of serum proteins to the polymeric matrix. However, in many locations individual cells could not be identified, probably due to the presence of secreted matrix elements. Cells grew through fibers and the thickness of the fibers increased. Cell clusters were observed both vertically and horizontally, covering several fibers. This suggests that cells grew due to a thin layer of electrosprayed fibers with large pore sizes.
Fig. 6. Cell distribution on different heights of the fibrous assembly at day 7. (A–C) The nuclei of cells on fibers at different heights in a single layer. (D) Overlayed image of fluorescence and light signals from cells and electrosprayed fibers in the single layer at 15.36 lm. (E–G) The nuclei of cells on fibers at different heights in the three layers. (E) Compilation of images in different planes showing the distribution of cells in three layers, best seen using 3-D spectacles.
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Fig. 7. Morphology of cells in a single layer of fibers shown by SEM.
3.4. Cell-glued 3-D scaffold To confirm whether three layers of fibers were attached after a 7 day cell culture, the sample was lifted by tweezers but the bottom layer of the sample was detached from the sample (Fig. 8A). Moreover, the sample disappeared while cutting with scissors because the complexity of the fibers and cells was still weak, mainly due to the fragile structure of the fibers (Fig. 8B). When cell cultures were continued up to 30 days, the three layers merged into a single structure, probably due to the regeneration of matrix elements and cell growth (Fig. 8C). Moreover, the shape of the 3-D scaffold was stable and tailorable without the deformation of the scaffold while and after the scaffold was cut by scissors (Fig. 8D). The samples were cut into small random pieces for further analysis by SEM and histology. The dry thickness of the three-layered assembly was 280 mm using SEM (not shown here). Clustered cells existed on the top and side of the 3-D scaffold. HFF-1 cells were distributed throughout the scaffold not only vertically but also horizontally, and this distribution supports the complex structure of cells and fibers without any mechanical damage even after cutting into the small pieces for H & E staining (Fig. 8E and F). Cells grew on the surface of the fibers. HFF-1s wrapped around a single fiber and elongated along the fiber. These cells attached adjacent cells growing on other nearby fibers not only vertically but also horizontally. The cells were distributed throughout in all dimensions of the scaffold without any distinguishable layers between the cells and fibers. This complex structure of fibers and cells in three layers glued three layers into one 3-D scaffold after 30 days, enabling the shape of the scaffold to be tailored and for it to be cut into small pieces without any mechanical damage. Thus, a cell-glued 3-D scaffold was fabricated using a thin layer of PCL fibers, with HFF1 in 15% serum-added medium after 30 days of cell culture, as a phenotype of a 3-D scaffold using electrosprayed fibers with a large pore size. 4. Discussion Biodegradable scaffolds are used to support and guide the ingrowth of cells and the scaffolding material eventually disappears, leaving only healthy tissue in a topologically required form. This
study has demonstrated the possibility of (i) developing very thin layers of fibers from different polymers, (ii) handling them in tissue culture conditions and (iii) building multiple layers of cells and fibers. Cells attach and distribute in 3-D synthetic polymer PCL. Since the technology is simple, the novel collector can be associated with existing techniques of electrospinning and applied to a number of other biomaterials. The thin layer of electrosprayed fibers is easy to handle without mechanical damage due to the support of the aluminum frame. Even when fibers were handled for the morphology study, the polymeric structures of the fibers remained intact, suggesting their ability to endure external stress. The aluminum foil provides a hard frame for the thin layers of the fibers to be picked up gently with tweezers, thus preventing the fibers from being destroyed. The pore size of fibers produced with the new collector plate is larger than of those produced with conventional collectors due to the decreased density of fibers. However, the diameter of fibers under the same conditions are similar, and can be manipulated by controlling the parameters of the solution (concentration of polymer solutions, solvent) and the process (flow rate, needle tip, distance between tip and collector, and high-voltage supply). Thus, larger pore sizes of fibers were generated while the diameter of fibers were controlled by using the collector. In other words, the innovative technique of the collector is easy to combine with the existing technology of electrospraying or electrospinning. This study opens a new window of opportunity to understand a number of cellular interactions in 3-D environment, mimicking physiological conditions. For example, a single layer can be visualized in a regular light microscope and the presence of fibers creates a rough architecture, similar to in a physiological environment. Thus, a single layer can also be used in cell culture to study the interactions in single cells in 3-D space. Traditional tissue culture conditions can be converted into 3-D cultures without significant effort. Thin layers can be placed serially to develop thick 3-D structures by adapting layer-by-layer assembly. One could build tissues to the required thickness layer-by-layer. SEM images of cells cultured on fibrous scaffolds fabricated using electrospinning showed that the cells can grow and proliferate within the fibrous scaffold. The cells both grew on the fibers and were intertwined by them. This novel collector overcame the
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Fig. 8. Cell-glued 3-D scaffolds. (A) Three layers of fibers after cell culture for 7 days. (B) Collapsing three layers while cutting after cell culture for 7 days (C). Cell-glued three layers of fibers after cell culture for 30 days. (D) Tailoring tissue-like materials after cell culture for 30 days. (E and F) Cell morphology after cell culture for 30 days using the top area and a cross-section of three layers stained with H & E for nuclei (red) and cytoplasm (dark pink).
usual barriers of electrosprayed and electrospun fibers. The majority of fibers made by the existing technology showed that the pore sizes between the fibers did not allow cells to infiltrate into the pores between and among the fibers. It was demonstrated that HFF-1 cells grew and proliferated when cultured on scaffolds made of thin layers of fibers fabricated by electrospraying using a novel collector. In addition, the single layer of electrospun fibers was expanded up to three layers. Further, the layer-by-layer assembly helps solve problems associated with the uniform seeding of cells into the porous structure, typically observed in many scaffolds synthesized by subtractive techniques, such as porogen-leaching and freeze-drying. Moreover, cells in the 3-D scaffold grew and became distributed throughout the sample, not only horizontally but also vertically, suggesting that the diffusion of the medium was sufficient for the cells to functionalize in all three layers. Thus, two core techniques of a thin layer of electrosprayed fibers with large pore sizes and layer-by-layer assembly can synergistically overcoming current barriers such as thicker tissue regeneration. In this study, PCL was used to demonstrate the concept of cell colonization on thin layers which do not have a cell-binding domain. Much of the cell adhesion is due to the interaction with the serum proteins, as cells were cultured in 15% serum-containing medium. To better understand the cellular interactions with fibers,
the changes in adsorption of serum protein need to be measured [25]. The cell-glued 3-D scaffolds demonstrated that cells in three layers were functional and distributed well throughout the sample. However, a thicker 3-D scaffold study using four or more layers will need to be undertaken to clarify the limitation of medium diffusion. When the limitation becomes clear, a new reactor design will be needed to allow cells to functionalize in thicker 3-D scaffolds. Not only that, the analysis of cellular activity will need to be extended to include other types and functionality; assembly and maturation of matrix elements in tissue regeneration plays a significant role in determining the biomechanics and the quality of the regenerated tissue. In addition, evaluating the alteration in cellular activity after immobilizing proteins containing cell adhesion domains could provide more information on the importance of fiber thickness and roughness. Recent advances in tissue regeneration and 3-D matrix physical properties such as hydrophilicity, stiffness [26], porosity [27], pore size and void fraction can affect cell morphology, attachment and function. Also, cellular activity is influenced by scaffold stiffness [28–30]. Cells show reduced spreading and disassembly of actin even when soluble adhesive ligands are present in weak gels [31,32]. The maximum tractional force generated by a cell can be as much as 10–15% of the substrate modulus [31]. Further, the rigidity of the scaffold may affect the
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formation of ECM, which, in turn, can affect cellular activity [33]. We performed bulk tensile properties. However, to understand the influence of scaffold stiffness, properties of individual fibers need to be analyzed using techniques such as nanoindentation. In addition, the influence of fiber curvature on cell colonization also needs to be analyzed. This study focused on using thin layers of electrosprayed fibers in tissue regeneration. This technique could also be used in numerous other applications, such as chemical and biochemical protection in defense and security, solar cells and fuel cells in sustainability, and membranes and filters in environmental engineering and biotechnology [34]. 5. Conclusion In summary, the novel collector plate allows the formation of thin layers of electrosprayed fibers with large pore sizes. The thin layer of electrosprayed fibers can be easily handled without mechanical damage because of the supporting structure. This collector is also versatile enough to be associated with existing techniques of electrospinning to manipulate mechanical and biological properties of novel fibers. This methodology overcame the major barrier of the electrospinning technique and its application for tissue regeneration. The novel fibers allow a single cell study on single fibers in a single layer, and in multilayers by using a layerby-layer technique. Furthermore, a cell-glued 3-D scaffold of the thin layer of electrosprayed fibers with large pore sizes will expand its use to other cells, fibers, tissues and organs. Acknowledgements Thanks to Dr. James Smay, Dr. Charlotte Ownby and Mr. Curtis Andrew for their help on using the scanning electron microscope, the confocal microscope and the H & E staining. Financial support was provided by the National Institutes of Health (1R21DK074858). Appendix A. Figures with essential colour discrimination Certain figures in this article, particularly Figures 1, 2, 4–6 and 8, are difficult to interpret in black and white. The full colour images can be found in the on-line version, at doi:10.1016/ j.actbio.2010.07.003. References [1] Langer R, Vacanti JP. Tissue engineering. Science 1993;260:920–6. [2] Persidis A. Tissue engineering. Nat Biotechnol 1999;17:508–10. [3] Vyavahare N, Ogle M, Schoen FJ, Zand R, Gloeckner DC, Sacks M, et al. Mechanisms of bioprosthetic heart valve failure: fatigue causes collagen denaturation and glycosaminoglycan loss. J Biomed Mater Res 1999;46:44–50. [4] Chvapil M. Collagen sponge: theory and practice of medical applications. J Biomed Mater Res 1977;11:721–41. [5] Oberpenning F, Meng J, Yoo JJ, Atala A. De novo reconstitution of a functional mammalian urinary bladder by tissue engineering. Nat Biotechnol 1999;17: 149–55. [6] Madihally SV, Matthew HW. Porous chitosan scaffolds for tissue engineering. Biomaterials 1999;20:1133–42.
[7] Mikos AG, Thorsen AJ, Czerwonka LA, Bao Y, Langer R, Winslow DN, et al. Preparation and characterization of poly(L-lactic acid) foams. Polymer 1994;35:1068–77. [8] Mooney DJ, Baldwin DF, Suh NP, Vacanti JP, Langer R. Novel approach to fabricate porous sponges of poly(D,L-lactic-co-glycolic acid) without the use of organic solvents. Biomaterials 1996;17:1417–22. [9] Wang YC, Ho CC. Micropatterning of proteins and mammalian cells on biomaterials. FASEB J 2004;8:8. [10] Hutmacher DW. Scaffolds in tissue engineering bone and cartilage. Biomaterials 2000;21:2529–43. [11] Morton WJ. Method of dispersing fluids. US Patent 1902;705691. [12] Sill TJ, von Recum HA. Electrospinning: applications in drug delivery and tissue engineering. Biomaterials 2008;29:1989–2006. [13] Shin H, Jo S, Mikos AG. Biomimetic materials for tissue engineering. Biomaterials 2003;24:4353–564. [14] Nisbet DR, Forsythe JS, Shen W, Finkelstein DI, Horne MK. Review paper: a review of the cellular response on electrospun nanofibers for tissue engineering. J Biomater Appl 2008: doi:0885328208099086. [15] Fibrous 3-dimensional scaffold via electrospinning for tissue regeneration and method for preparing the same. USPTO 2008;0233162. [16] Stankus JJ, Soletti L, Fujimoto K, Hong Y, Vorp DA, Wagner WR. Fabrication of cell microintegrated blood vessel constructs through electrohydrodynamic atomization. Biomaterials 2007;28:2738–46. [17] Mondalek FG, Lawrence BJ, Kropp BP, Grady BP, Fung KM, Madihally SV, et al. The incorporation of poly(lactic-co-glycolic) acid nanoparticles into porcine small intestinal submucosa biomaterials. Biomaterials 2008;29:1159–66. [18] Sarasam A, Madihally SV. Characterization of chitosan–polycaprolactone blends for tissue engineering applications. Biomaterials 2005;26:5500–8. [19] Pok SW, Wallace KN, Madihally SV. In vitro characterization of polycaprolactone matrices generated in aqueous media. Acta Biomater 2010;6:1061–8. [20] Madibally SV, Solomon V, Mitchell RN, Van De Water L, Yarmush ML, Toner M. Influence of insulin therapy on burn wound healing in rats. J Surg Res 2003;109:92–100. [21] Gupta P, Wilkes GL. Some investigations on the fiber formation by utilizing a side-by-side bicomponent electrospinning approach. Polymer 2003;44: 6353–9. [22] Hellmann C, Belardi J, Dersch R, Greiner A, Wendorff JH, Bahnmueller S. High precision deposition electrospinning of nanofibers and nanofiber nonwovens. Polymer 2009;50:1197–205. [23] Kidoaki S, Kwon IK, Matsuda T. Mesoscopic spatial designs of nano- and microfiber meshes for tissue-engineering matrix and scaffold based on newly devised multilayering and mixing electrospinning techniques. Biomaterials 2005;26:37–46. [24] Varesano A, Carletto RA, Mazzuchetti G. Experimental investigations on the multi-jet electrospinning process. J Mater Process Technol 2009;209:5178–85. [25] Tillman J, Ullm A, Madihally SV. Three-dimensional cell colonization in a sulfate rich environment. Biomaterials 2006;27:5618–26. [26] Balgude AP, Yu X, Szymanski A, Bellamkonda RV. Agarose gel stiffness determines rate of DRG neurite extension in 3D cultures. Biomaterials 2001;22:1077–84. [27] Dar A, Shachar M, Leor J, Cohen S. Optimization of cardiac cell seeding and distribution in 3D porous alginate scaffolds. Biotechnol Bioeng 2002;80: 305–12. [28] Zaleskas JM, Kinner B, Freyman TM, Yannas IV, Gibson LJ, Spector M. Growth factor regulation of smooth muscle actin expression and contraction of human articular chondrocytes and meniscal cells in a collagen–GAG matrix. Exp Cell Res 2001;270:21–31. [29] Lee CR, Grodzinsky AJ, Spector M. The effects of cross-linking of collagen– glycosaminoglycan scaffolds on compressive stiffness, chondrocyte-mediated contraction, proliferation and biosynthesis. Biomaterials 2001;22:3145–54. [30] Sieminski AL, Hebbel RP, Gooch KJ. The relative magnitudes of endothelial force generation and matrix stiffness modulate capillary morphogenesis in vitro. Exp Cell Res 2004;297:574–84. [31] Lo CM, Wang HB, Dembo M, Wang YL. Cell movement is guided by the rigidity of the substrate. Biophys J 2000;79:144–52. [32] Pelham Jr RJ, Wang Y. Cell locomotion and focal adhesions are regulated by substrate flexibility. Proc Natl Acad Sci USA 1997;94:13661–5. [33] Wozniak MA, Modzelewska K, Kwong L, Keely PJ. Focal adhesion regulation of cell behavior. Biochim Biophys Acta 2004;1692:103–19. [34] Ramakrishna S, Fujihara K, Teo W-E, Yong T, Ma Z, Ramaseshan R. Electrospun nanofibers: solving global issues. Mater Today 2006;9:40–50.
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Acta Biomaterialia journal homepage: www.elsevier.com/locate/actabiomat
Three-dimensional scaffold of electrosprayed fibers with large pore size for tissue regeneration Jong Kyu Hong, Sundararajan V. Madihally * School of Chemical Engineering, Oklahoma State University, 423 Engineering North, Stillwater, OK 74078, USA
a r t i c l e
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Article history: Received 22 April 2010 Received in revised form 30 June 2010 Accepted 2 July 2010 Available online 8 July 2010 Keywords: Electrospraying PCL Gelatin Tissue regeneration Porous structure
a b s t r a c t The regeneration of tissues using biodegradable porous scaffolds has been intensely investigated. Since electrospinning can produce scaffolds mimicking nanofibrous architecture found in the body, it has recently gained widespread attention. However, a major problem is the lack of pore size necessary for infiltration of cells into the layers below the surface, restricting cell colonization to the surfaces only. This study describes a novel twist to the traditional electrospinning technology: specifically, collector plates are designed which allow the formation of very thin layers with pore sizes suitable for cell infiltration. The thin samples could be handled without mechanically damaging the structure and could be transferred into cell culture. These thin layers were stacked layer-by-layer to develop thick structures. Thirty day cultures of fibroblasts show attachment and spreading of cells in every layer. This concept is useful in regenerating thick tissues with uniformly distributed cells and others in in vitro cell culture. Ó 2010 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.
1. Introduction Tissue engineering or regeneration focuses on restoring, maintaining and enhancing tissue and organ function [1]. In tissue engineering, biodegradable scaffolds are used to support and guide cells to proliferate, organize and produce their own extracellular matrix (ECM) [2] so that, when the scaffolding material eventually disappears, only the necessary healthy tissue is left in a topologically required form [3]. A biomimic matrix for growing cells is a fundamental element for tissue regeneration. Templates generated from natural [4] and synthetic polymers or after removing the cellular components from xenogeneic tissues [5] have been used to support and guide the in-growth of cells. Synthesizing scaffolds using techniques such as controlled rate freezing and lyophilization [6], the porogen-leaching technique [7], the gas-foaming technique [8], microfabrication [9] and free-form printing [10] has attracted increasing attention. Although each technique has advantages and disadvantages, the electrospinning technique has recently emerged as a novel technique for tissue regeneration because it allows fabrication of nano- and micrometer size fibers, which are very similar to fibers in the natural extracellular environment. The process of fabricating nano- to micrometer synthetic fibers with non-woven structures using electrostatic forces, called electrospinning (electrospray and spinning), has been known for over * Corresponding author. Tel.: +1 405 744 9115; fax: +1 405 744 6338. E-mail addresses: [email protected], [email protected] (S.V. Madihally).
100 years. Although electrospinning technology was first patented in the US in 1902 [11], this technology was latent until the era of nanoscience and nanotechnology in the 1990s. The electrospinning process is versatile and its productivity is higher than other methods of fabrication of nanofibers. With a suitable polymer and appropriate solvent system, fiber size can be controlled (from 40 nm to 10 lm) by manipulating the process parameters. Since the technology allows the possibility of tailoring the mechanical and biological properties, there significant efforts have been made to adapt the technology for use in tissue regeneration [12,13]. Most soluble polymers with a high molecular weight can be electrosprayed (fibers collected on a stationary collector plate) or electrospun. Nanofibers can be made of natural and synthetic polymers, or polymer blends. A major problem in electrospinning technology is the lack of generating structural features necessary for building three-dimensional (3-D) tissues [12,14]. The pore sizes are less than 10 lm, while human cells are typically greater than 10 lm in size [15]. Hence, cell growth is restricted to the surface only. The reduced pore size is primarily due to multiple layers of fibers deposited to obtain thicker structures that withstand mechanical handling in subsequent steps. Addressing the cell seeding problem by depositing both the polymer and the cells simultaneously is a recent advancement [16]. However, this approach is not practical as cells will be exposed to (i) toxic organic solvents used in the process, (ii) high shear rates induced by the flow of fluids through narrow nozzles and (iii) sub-optimal environmental conditions during manufacturing. These factors affect cell viability and growth function tissue growth. Due to reduced pore size and restricted cell
1742-7061/$ - see front matter Ó 2010 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.actbio.2010.07.003
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infiltration, it is not possible to regenerate tissues of a thickness suitable for clinical transplantation. In this study, we introduce an innovative electrospraying technique with a novel collector plate which allows the formation of very thin layers of micro- and nanofibers. Due to reduced thickness, pore size (60 lm) of the formed layers is suitable for mammalian cell infiltration. We performed the fibroblast cell culture of multiple layers using layer-by-layer assembly: place one layer of thin fibers, seed cells and repeat. We show 3-D scaffolds of thin layers of electrosprayed fibers in which human fibroblasts are growing not only horizontally but also vertically due to the large pore sizes of the electrosprayed fibers. The implications of producing 3-D scaffolds using the layer-by-layer technique can be extrapolated to other cells, fibers, tissues and organs, and could become a phenotype of 3-D scaffolds in tissue regeneration. 2. Materials and methods 2.1. Materials Polycaprolactone (PCL; Mn = 80,000) and gelatin type A were purchased from Sigma (St. Louis, MO) and PCL (Mw = 43,000–50,000) was obtained from Polysciences (Warrington, PA). Chloroform, methanol and acetic acid were purchased from Pharmco (Brookfield, CT). For cell culture study, human foreskin fibroblast (HFF-1) was obtained from the American Type Culture Collection (Walkersville, MD). Dulbecco’s modified Eagle’s medium (DMEM), glucose and amphotericin B were obtained from Sigma (St. Louis, MO). Fetal
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bovine serum (FBS), Alexa Fluor 546 and 40 ,6-diamidino-2-phenylindole (DAPI) were purchased from Invitrogen (Carlsbad, CA). TrypLE Express, penicillin–streptomycin and L-glutamine were obtained from Gibco (Grand Island, NY) and sodium bicarbonate was purchased from EMD (Gibbstown, NJ). 2.2. Fabrication of the thin layer of electrosprayed fibers Electrospraying apparatus consisted of a syringe pump (74,900 series, Cole-Parmer Instrument Company, Vernon Hills, IL), a BD 10 ml syringe (Luer-Lok Tip; Becton Dickinson and Company, Franklin Lakes, NJ), a needle tip, a high-voltage power supply (ES30P-5W/DAM, Gamma high Voltage Research, Ormond Beach, FL), earth grounding and a collector plate (Fig. 1A). The novel collector had four holes (diameter, 1.9 cm and depth 1.2 cm) in a wooden frame (3 10 2 cm) wrapped with aluminum foil (Fig. 1E). A conventional collector plate that was the same size as the novel collector but without a hole was also wrapped with aluminum foil. Electrosprayed fibers were fabricated at 40 ml h1 and 20 kV. For microsized fibers, 20% (w/v) PCL (Mw = 43,000–50,000) dissolved in a mixture solvent of chloroform and methanol (mixing ratio 9 to 1) was sprayed using a 14G needle. The distance of the collector plate was 12 cm from the tip of the needle. For nanosized fibers, 10% (w/v) PCL dissolved in a mixture solvent of chloroform and methanol (mixing ratio 9 to 1) was sprayed using a 26G needle. The distance of the collector plate was 15 cm from the tip of the needle. Deposit volume of the solution was 0.75 ml. Gelatin fibers were formed using 20% (w/v) gelatin dissolved in acetic acid and
Fig. 1. Novel electrospray set-up. (A) Schematic representation of conventional electrospray set-up. (B) Schematic representation of novel electrospray set-up. (C) Photograph of conventional electrospray set-up. (D) Photograph of novel electrospray set-up. (E) Photograph of the novel collector.
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distilled water (mixing ratio 7 to 3). Flow rate, needle size, highvoltage supply and distance between the needle tip and the collector were 10 ml h1, 20G, 10 kV and 8 cm, respectively. For simultaneous and sequential deposit, 20% w/v PCL (Mn = 80,000) was prepared with a solvent mixture of chloroform and methanol (mixing ratio 9 to 1). In the simultaneous and the sequential processes, fibers were formed at the same condition described for gelatin fiber formation using the novel collector. 2.3. Microstructure characterization Fig. 2. Schematic of the process of cell seeding using layer-by-layer assembly.
Samples were analyzed using a JEOL 6360 electron microscope (Jeol USA Inc., Peabody, MA) at an accelerated voltage of 9–20 kV, similar to our previous publications [17]. In brief, samples were attached to an aluminum stub using a conductive graphite glue (Ted Pella Inc., Redding, CA) and sputter-coated with gold for 1 min before scanning electron microscopy (SEM) study. Along with the obtained SEM images, fiber diameters, pore size and shape factors of pores (4p area/perimeter2; the closer the number is to 1, the closer the cell shape is to a circle) were quantified using Sigma Scan Pro (SPSS Science, Chicago, IL). More than 35 points of fibers and pores were analyzed. 2.4. Mechanical test Microsize fibers were fabricated using a collector plate (10 3.5 2 cm) with a rectangular hole (5 1 1 cm) and an additional flat surface area or peripheral area of a rectangular hole, resulting in two rectangular holes (one with a flat surface and the other without). Fibers accumulated on the flat surface were used as samples for conventional fibers while the fibers collected in the hole were used as samples for novel fibers. Tensile testing was performed at room temperature with dry conditions using the method described previously [18]. In brief, 25 mm (width) 10 mm (length) rectangular strips were cut from the novel and conventional samples and strained to break at a constant crosshead speed of 10 mm min1 using INSTRON 5542 tension machine (INSTRON, Canton, MA). 2.5. Cell culture HFF-1 was cultured in DMEM supplemented with 4 mM L-glutamine, 4.5 g l1 glucose, 1.5 g l1 sodium bicarbonate, 100 U ml1 penicillin–streptomycin, 2.5 mg ml1 amphotericin B and 15% FBS. The cells were maintained in a CO2 incubator at 37 °C. The medium was changed every 2 days. Before cell seeding, cells were detached with 2% TrypLE Express, centrifuged and suspended in serum-added medium. Microsize fibers were fabricated using 20% w/v of PCL (Mw = 43,000) in chloroform/methanol (ratio 9 to 1) and the novel collector. Flow rate, needle size, high-voltage supply and distance between the needle tip and the collector were 10 ml h1, 20G, 10 kV and 8 cm, respectively. The deposit volume was 0.75 ml. Fibers on the aluminum frame were sterilized using UV light for 4 h, immersed in ethanol for 4 h and rinsed with phosphate-buffered saline (PBS) three times. The thin layers were soaked in serumadded medium for 4 h. The cell culture plates were treated with albumin to minimize cell adhesion to the tissue culture plastic surface prior to insertion of a thin layer of fibers (Fig. 2). Then 30,000 cells in 100 ll were seeded onto five points of fibers in a single layer of the aluminum frame. For three-layer cultures, the cell culture in the single layer was repeated three times using the layer-by-layer technique: place one layer of the sample, seed cells on five points of the sample and repeat the process of sample loading and cell seeding twice. Ten
minutes after cell seeding, 2 and 3 ml of medium were added to six-well plates for single and triple layers, respectively. 2.6. Evaluating of cell distribution and organization At 1, 4 and 7 days after cell culture, the sample was fixed with 3.7% formaldehyde, rinsed with PBS and immersed in ethanol at 20 °C. The sample was then stained with 2 ml of Alexa phalloidin (0.66 lM) for cytoskeletal actin and counterstained with 2 ml of DAPI (30 nmol) for nuclei of cells, respectively [19]. The samples were first observed under an inverted fluorescence microscope (Nikon TE2000, Melville, NY). Digital micrographs were collected using a CCD camera. The data were treated using Corel Paint Shop Pro Photo X2 to modulate the brightness, contrast and clarification. Also, 3-D images of cells on fibers in the single and triple layers were collected using a confocal microscope (Leica TCS SP II, Heidelberg, Germany). Image stacks of both single and triple layers along the z-axis were collected at a step size of 1.282 lm for the singlelayer cultures and 4.271 lm for the three-layer cultures. The observed thickness of the z-axis for single and triple layers were 30.77 and 89.70 lm, respectively. At each step, the two fluorescent signals of nuclei and skeletal actin and one light signal of fibers were collected. Then fluorescent and light signals of cells on fibers in the single layer at 15.36 lm along the z-axis were overlapped. To obtain a 3-D hologram, fluorescence signals of cells at each step were merged in the three-layer sample. Samples were also analyzed using SEM to evaluate the distribution of fibers and cells. For this purpose, the samples were dried using ethanol and analyzed similar to the fibers described above (Section 2.3). 2.7. Histology assay After 30 days of cell culture, the samples formed using single and triple layers of fibers were tailored using tweezers and scissors. The aluminum frames of the samples were removed completely. Random parts of the samples were fixed in 3.7% formaldehyde, dried through an ethanol gradient and embedded in paraffin. The samples were cut into 4 lm and stained with hematoxylin and eosin (H & E) for histology analysis [20]. 3. Results 3.1. Fabrication of the thin layer of highly porous fibers A conventional electrospray set-up consists of a syringe pump, a syringe and a needle of appropriate size, a high-voltage power supply, earth grounding and a stationary collector plate (Fig. 1A). The collector plate is a flat conducting plate where the polymer fibers accumulate. After a certain elution volume, the fibers are removed and used in the required application. To remove the material, larger volumes are deposited, which leads to fibrous structures with limited pore size.
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We questioned whether the fibers could be accumulated by creating a hole in the collector plate. For this purpose, different size holes were created in wood, plastic or glass, and wrapped around with a conductive aluminum foil (Fig. 1B), which can be autoclaved when sterilization is necessary. The collector plate was designed with the intention of using it as a support structure while transporting and subsequent handling. Experiments performed under the same conditions as the traditional collector plate showed the presence of fibers in the holed area, suggesting the possibility of forming thin layers. Different shapes, such as circle, hexagon and rectangles, of various sizes (up to 10 cm) were tested. These results showed the possibility of obtaining thin layers. Since the aluminum foil could be simply peeled off from the core structure, it is also easy for the thin layer to be handled without mechanical damage from external stress. The absence of a continuous conducting medium could alter the electrical field. Hence, we questioned how the distribution of fibers would be altered and whether the fibers formed would have a different architecture. Similar to other reports, fibers generated by the conventional plate showed multiple layers of fibers (Fig. 3A) under SEM. However, few layers of fibers were observed in the open area of the new collector plate (Fig. 3B), while multiple layers were observed on the sheet. Many fiber morphologies appeared smooth and similar between the two conditions, except in the case of a large fiber; in the conventional plate (Fig. 3A), many fibers showed pores on their surface. To test whether nanosize fibers could be obtained using the new collector plate, processing parameters were optimized by manipulating the flow rate of the solution, the strength of voltage, and the distance between the needle tip and the collector. The conventional collector plate showed multilay-
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ered nanofibers (Fig. 3C). Similar nanofibers were collected on the new collector plate with a single layer (Fig. 3D), suggesting the applicability of this collector plate to other conditions. To test whether the new collector plate is applicable to other polymeric systems, the formation of fibers using naturally derived gelatin was tested. The results showed that the gelatin fibers accumulated similar to the PCL fibers (Fig. 3E). Others have also used sequential (exchanging the syringes containing two different polymeric solutions in sequence) and simultaneous (using two syringes containing two different polymeric solutions at the same time) processes of electrospraying to deposit two different polymers for controlling the microarchitecture [21–24]. To test this possibility, PCL and gelatin were deposited sequentially and simultaneously. The results showed (Fig. 3F) that thin layers of fibers of different polymers could be collected on the novel collector plate. To understand the effect of hydration on the fibers, formed fibers were observed under light microscopy before and after hydration. Upon hydration in PBS, the PCL fiber structure remained the same. However, gelatin fibers dissolved completely in PBS as they were not stabilized by any cross-linker. When composite structures were hydrated, PCL fibers remained intact even after 30 or more days. However, the fibers made of PCL and gelatin were entangled and collapsed within 15 min because gelatin dissolved in PBS. Thus, one could easily understand the alterations in fibrous architecture using these thin layers. 3.2. Fiber characterization To determine the fiber size and pore size, fibers were generated under the same condition of solution (concentration of solution)
Fig. 3. Morphology of fibers using novel and conventional collectors. Microfibers made of PCL (Mw = 43,000) with (A) a conventional collector and (B) a novel collector under the same conditions. Nanofibers made of PCL (Mw = 43,000) with (C) a conventional collector and (D) a novel collector under the same conditions. (E) Fibers made of gelatin type A using the novel collector. (F) Hybrid fibers made of microsize PCL (Mn = 80,000) fibers and nanosize gelatin type A fibers in sequential process using the novel collector.
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and process parameters (flow rate, needle size, distance between the needle tip and the collector, and high-voltage supply) using the novel and the conventional collector plates. Quantification of scanning electron micrographs showed that the new collector plate does not affect the fiber size and the shape of the pores in the scaffolds (Fig. 4A). Even when nanofibers were formed under other conditions, no significant difference was obtained. Thus, this new collector plate can be easily adapted to various other polymers in conjunction with the intended application. Controlling fiber sizes is also possible using appropriate solvents and the distance of the collector plate. This suggests that the technology is versatile and can be easily integrated into existing technologies. The shape factor (Fig. 4B) of pores formed due to random distribution of fibers was similar in the two cases. Pore size (Fig. 4C) of the scaffold formed with the new collector plate showed significant increase due to the decreased density of fibers deposited in the open area. However, under the same conditions of solution and process parameters, the diameters of fibers generated by the novel and conventional fibers were quite similar. The pore size of fiber produced with the novel collector is much greater than those produced with the conventional one. Collected fibrous structures at the same condition for the same time were tested under tensile loading in the dry state at room temperature. Load–extension curves (Fig. 4D) showed that the extension of novel fibers was twice as long as that of conventional fibers; novel fibers showed 250% strain, whereas the conventional fibers showed 100% strain. However, the load-carrying capacity of the fibrous structures was nearly half that of the conventional fibers. These differences could be attributed to the decreased density of fibers in the novel collector plate; due to free space between the fibers on the sample, thin layers can stretch but cannot carry too much load.
3.3. Developing 3-D colonized cells To test whether cells can be cultured on these thin layers, porous structures formed along with the aluminum foil were inserted onto albumin-coated culture plates (to minimize cell adhesion to the tissue culture plastic) seeded with HFF-1 cells. For evaluating layer-by-layer assembly, the process of inserting the thin layer and seeding cells was done three times (Fig. 2). The entire assembly was cultured for 7 days in serum-containing medium with routine monitoring under light microscopy. An increase in the number of cells was observed on fibers in both the single and triple layers. Further, many cells in both the single and triple layers appeared in clusters at the initial time point, but showed spreading and a spindle shape. By day 7, a number of pores were covered with the regenerated components. To assess the cellular components, they were stained for cytoskeletal actin and counterstained for nucleus using DAPI. To avoid background fluorescence signal from cells on the surface of the cell culture dish, the samples were moved into new dishes after the staining and washing. Fluorescent images (Fig. 5) in single or triple layers showed a qualitative improvement in the cells up to 7 days. Moreover, cells were individually distributed and surrounded by matrix elements, suggesting that the cells are functional. In addition, some cells were focused in the plane of view and others were unfocused, as they were attached to different layers of fibers. To better understand the distribution of cells in three dimensions, confocal microscopic analysis was performed using a step size of 1.2821 lm. Reconstructed confocal images of three layers showed the presence of cells in every 89.7 lm thick layer of the three-layer culture (Fig. 6). More cells appear in a single layer culture as they are distributed in a narrow thickness. To assess the distribution of cells in relation to fibers, a light microscopy image
Fig. 4. Comparison of fibrous layer characteristics. (A) Fiber diameters, with error bars corresponding to the standard deviation (n = 50). (B) Shape factor, with error bars corresponding to the standard deviation (n = 50). (C) Box plot showing the distribution of pore size with 10th, 25th, 50th, 75th and 90th percentiles and the mean value (thick line within each box). Values that were outside the 95th and 5th percentiles were treated as outliers. (D) The load–extension behavior.
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Fig. 5. Cell colonization in thin layers using layer-by-layer assembly. (A–C) Fluorescent micrographs of cells on single layer of fibers. (D–F) Fluorescent micrographs of cells on three layers of fibers. The samples were stained with Alexa phalloidin for cytoskeletal actin (red) and counterstained with DAPI for nuclei (blue) of cells. The scale bar corresponds to 50 lm.
was overlayed with fluorescent images in the same plane. The obtained micrograph showed that cells were surrounded by fibers and attached to the fibers, particularly in pores which were not too large relative to the size of the cells. Note that Fig. 6H is a 3-D image which can be better visualized using 3-D spectacles. This suggests that cells are distributed continuously in three dimensions. Samples were also evaluated using SEM to determine the distribution of cells within the fibers. These results revealed (Fig. 7A) that the cells on all fibers were attached to the fibers and entangled
by them. Cells attached and spread throughout the matrix. In some locations, cells showed numerous lamellipodia and filopodia with anchorage points to the fibers (Fig. 7B), probably via the adhesion of serum proteins to the polymeric matrix. However, in many locations individual cells could not be identified, probably due to the presence of secreted matrix elements. Cells grew through fibers and the thickness of the fibers increased. Cell clusters were observed both vertically and horizontally, covering several fibers. This suggests that cells grew due to a thin layer of electrosprayed fibers with large pore sizes.
Fig. 6. Cell distribution on different heights of the fibrous assembly at day 7. (A–C) The nuclei of cells on fibers at different heights in a single layer. (D) Overlayed image of fluorescence and light signals from cells and electrosprayed fibers in the single layer at 15.36 lm. (E–G) The nuclei of cells on fibers at different heights in the three layers. (E) Compilation of images in different planes showing the distribution of cells in three layers, best seen using 3-D spectacles.
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Fig. 7. Morphology of cells in a single layer of fibers shown by SEM.
3.4. Cell-glued 3-D scaffold To confirm whether three layers of fibers were attached after a 7 day cell culture, the sample was lifted by tweezers but the bottom layer of the sample was detached from the sample (Fig. 8A). Moreover, the sample disappeared while cutting with scissors because the complexity of the fibers and cells was still weak, mainly due to the fragile structure of the fibers (Fig. 8B). When cell cultures were continued up to 30 days, the three layers merged into a single structure, probably due to the regeneration of matrix elements and cell growth (Fig. 8C). Moreover, the shape of the 3-D scaffold was stable and tailorable without the deformation of the scaffold while and after the scaffold was cut by scissors (Fig. 8D). The samples were cut into small random pieces for further analysis by SEM and histology. The dry thickness of the three-layered assembly was 280 mm using SEM (not shown here). Clustered cells existed on the top and side of the 3-D scaffold. HFF-1 cells were distributed throughout the scaffold not only vertically but also horizontally, and this distribution supports the complex structure of cells and fibers without any mechanical damage even after cutting into the small pieces for H & E staining (Fig. 8E and F). Cells grew on the surface of the fibers. HFF-1s wrapped around a single fiber and elongated along the fiber. These cells attached adjacent cells growing on other nearby fibers not only vertically but also horizontally. The cells were distributed throughout in all dimensions of the scaffold without any distinguishable layers between the cells and fibers. This complex structure of fibers and cells in three layers glued three layers into one 3-D scaffold after 30 days, enabling the shape of the scaffold to be tailored and for it to be cut into small pieces without any mechanical damage. Thus, a cell-glued 3-D scaffold was fabricated using a thin layer of PCL fibers, with HFF1 in 15% serum-added medium after 30 days of cell culture, as a phenotype of a 3-D scaffold using electrosprayed fibers with a large pore size. 4. Discussion Biodegradable scaffolds are used to support and guide the ingrowth of cells and the scaffolding material eventually disappears, leaving only healthy tissue in a topologically required form. This
study has demonstrated the possibility of (i) developing very thin layers of fibers from different polymers, (ii) handling them in tissue culture conditions and (iii) building multiple layers of cells and fibers. Cells attach and distribute in 3-D synthetic polymer PCL. Since the technology is simple, the novel collector can be associated with existing techniques of electrospinning and applied to a number of other biomaterials. The thin layer of electrosprayed fibers is easy to handle without mechanical damage due to the support of the aluminum frame. Even when fibers were handled for the morphology study, the polymeric structures of the fibers remained intact, suggesting their ability to endure external stress. The aluminum foil provides a hard frame for the thin layers of the fibers to be picked up gently with tweezers, thus preventing the fibers from being destroyed. The pore size of fibers produced with the new collector plate is larger than of those produced with conventional collectors due to the decreased density of fibers. However, the diameter of fibers under the same conditions are similar, and can be manipulated by controlling the parameters of the solution (concentration of polymer solutions, solvent) and the process (flow rate, needle tip, distance between tip and collector, and high-voltage supply). Thus, larger pore sizes of fibers were generated while the diameter of fibers were controlled by using the collector. In other words, the innovative technique of the collector is easy to combine with the existing technology of electrospraying or electrospinning. This study opens a new window of opportunity to understand a number of cellular interactions in 3-D environment, mimicking physiological conditions. For example, a single layer can be visualized in a regular light microscope and the presence of fibers creates a rough architecture, similar to in a physiological environment. Thus, a single layer can also be used in cell culture to study the interactions in single cells in 3-D space. Traditional tissue culture conditions can be converted into 3-D cultures without significant effort. Thin layers can be placed serially to develop thick 3-D structures by adapting layer-by-layer assembly. One could build tissues to the required thickness layer-by-layer. SEM images of cells cultured on fibrous scaffolds fabricated using electrospinning showed that the cells can grow and proliferate within the fibrous scaffold. The cells both grew on the fibers and were intertwined by them. This novel collector overcame the
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Fig. 8. Cell-glued 3-D scaffolds. (A) Three layers of fibers after cell culture for 7 days. (B) Collapsing three layers while cutting after cell culture for 7 days (C). Cell-glued three layers of fibers after cell culture for 30 days. (D) Tailoring tissue-like materials after cell culture for 30 days. (E and F) Cell morphology after cell culture for 30 days using the top area and a cross-section of three layers stained with H & E for nuclei (red) and cytoplasm (dark pink).
usual barriers of electrosprayed and electrospun fibers. The majority of fibers made by the existing technology showed that the pore sizes between the fibers did not allow cells to infiltrate into the pores between and among the fibers. It was demonstrated that HFF-1 cells grew and proliferated when cultured on scaffolds made of thin layers of fibers fabricated by electrospraying using a novel collector. In addition, the single layer of electrospun fibers was expanded up to three layers. Further, the layer-by-layer assembly helps solve problems associated with the uniform seeding of cells into the porous structure, typically observed in many scaffolds synthesized by subtractive techniques, such as porogen-leaching and freeze-drying. Moreover, cells in the 3-D scaffold grew and became distributed throughout the sample, not only horizontally but also vertically, suggesting that the diffusion of the medium was sufficient for the cells to functionalize in all three layers. Thus, two core techniques of a thin layer of electrosprayed fibers with large pore sizes and layer-by-layer assembly can synergistically overcoming current barriers such as thicker tissue regeneration. In this study, PCL was used to demonstrate the concept of cell colonization on thin layers which do not have a cell-binding domain. Much of the cell adhesion is due to the interaction with the serum proteins, as cells were cultured in 15% serum-containing medium. To better understand the cellular interactions with fibers,
the changes in adsorption of serum protein need to be measured [25]. The cell-glued 3-D scaffolds demonstrated that cells in three layers were functional and distributed well throughout the sample. However, a thicker 3-D scaffold study using four or more layers will need to be undertaken to clarify the limitation of medium diffusion. When the limitation becomes clear, a new reactor design will be needed to allow cells to functionalize in thicker 3-D scaffolds. Not only that, the analysis of cellular activity will need to be extended to include other types and functionality; assembly and maturation of matrix elements in tissue regeneration plays a significant role in determining the biomechanics and the quality of the regenerated tissue. In addition, evaluating the alteration in cellular activity after immobilizing proteins containing cell adhesion domains could provide more information on the importance of fiber thickness and roughness. Recent advances in tissue regeneration and 3-D matrix physical properties such as hydrophilicity, stiffness [26], porosity [27], pore size and void fraction can affect cell morphology, attachment and function. Also, cellular activity is influenced by scaffold stiffness [28–30]. Cells show reduced spreading and disassembly of actin even when soluble adhesive ligands are present in weak gels [31,32]. The maximum tractional force generated by a cell can be as much as 10–15% of the substrate modulus [31]. Further, the rigidity of the scaffold may affect the
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formation of ECM, which, in turn, can affect cellular activity [33]. We performed bulk tensile properties. However, to understand the influence of scaffold stiffness, properties of individual fibers need to be analyzed using techniques such as nanoindentation. In addition, the influence of fiber curvature on cell colonization also needs to be analyzed. This study focused on using thin layers of electrosprayed fibers in tissue regeneration. This technique could also be used in numerous other applications, such as chemical and biochemical protection in defense and security, solar cells and fuel cells in sustainability, and membranes and filters in environmental engineering and biotechnology [34]. 5. Conclusion In summary, the novel collector plate allows the formation of thin layers of electrosprayed fibers with large pore sizes. The thin layer of electrosprayed fibers can be easily handled without mechanical damage because of the supporting structure. This collector is also versatile enough to be associated with existing techniques of electrospinning to manipulate mechanical and biological properties of novel fibers. This methodology overcame the major barrier of the electrospinning technique and its application for tissue regeneration. The novel fibers allow a single cell study on single fibers in a single layer, and in multilayers by using a layerby-layer technique. Furthermore, a cell-glued 3-D scaffold of the thin layer of electrosprayed fibers with large pore sizes will expand its use to other cells, fibers, tissues and organs. Acknowledgements Thanks to Dr. James Smay, Dr. Charlotte Ownby and Mr. Curtis Andrew for their help on using the scanning electron microscope, the confocal microscope and the H & E staining. Financial support was provided by the National Institutes of Health (1R21DK074858). Appendix A. Figures with essential colour discrimination Certain figures in this article, particularly Figures 1, 2, 4–6 and 8, are difficult to interpret in black and white. The full colour images can be found in the on-line version, at doi:10.1016/ j.actbio.2010.07.003. References [1] Langer R, Vacanti JP. Tissue engineering. Science 1993;260:920–6. [2] Persidis A. Tissue engineering. Nat Biotechnol 1999;17:508–10. [3] Vyavahare N, Ogle M, Schoen FJ, Zand R, Gloeckner DC, Sacks M, et al. Mechanisms of bioprosthetic heart valve failure: fatigue causes collagen denaturation and glycosaminoglycan loss. J Biomed Mater Res 1999;46:44–50. [4] Chvapil M. Collagen sponge: theory and practice of medical applications. J Biomed Mater Res 1977;11:721–41. [5] Oberpenning F, Meng J, Yoo JJ, Atala A. De novo reconstitution of a functional mammalian urinary bladder by tissue engineering. Nat Biotechnol 1999;17: 149–55. [6] Madihally SV, Matthew HW. Porous chitosan scaffolds for tissue engineering. Biomaterials 1999;20:1133–42.
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